1. Field of the Invention
The present invention relates to the fields of microbiology and fermentation technology. The present invention further relates to the use of novel bacterial strains, ingredients and processes useful for the fermentative production of 2-keto-L-gulonic acid from D-sorbitol.
2. Related Art
2-Keto-L-gulonic acid (xe2x80x9c2-KLGxe2x80x9d) is a significant intermediate in the preparation of L-ascorbic acid (vitamin C), an essential nutrient. 2-KLG has been synthesized in the past on an industrial scale using the Reichstein method (Helvetica Chimica Acta 17:311 (1934)). This method, however, has a number of disadvantages for commercial application, including the use of large quantities of solvents and the involvement of a number of complex reaction steps.
Accordingly, as an alternative to the Reichstein method, a number of processes employing one or more microorganisms have been developed to produce 2-KLG by fermentation. U.S. Pat. No. 2,421,611, for example, discloses a method involving microbial oxidation of D-glucose to 5-keto-D-gluconic acid, followed by chemical or microbial reduction to L-idonic acid and subsequent microbial oxidation to 2-KLG. U.S. Pat. No. 3,998,697 discloses a similar process involving the microbial oxidation of D-glucose to 2,5-diketo-D-gluconic acid, followed by microbial reduction to 2-KLG.
These methods, however, also suffer from a number of disadvantages that reduce their usefulness in commercial production of 2-KLG. For example, the chemical reduction steps in these methods (i.e. the reduction of 5-keto-D-gluconic acid to L-idonic acid and 2,5-diketo-D-gluconic acid to 2-KLG) are accompanied by problems with controlling the stereochemistry of reduction (thus producing D-gluconic acid and 2-keto-D-gluconic acid, respectively, as byproducts) which, in turn, reduces the yield of 2-KLG. Alternatively, when this reduction is performed by one or more microorganisms, excess sugar is required to provide an energy source for the reduction, which also reduces the yield of 2-KLG.
In view of these problems, alternate pathways have been employed for the fermentative production of 2-KLG. A number of processes have been developed which involve oxidation of L-sorbose to 2-KLG via a sorbosone intermediate that employ a wide range of microorganisms from the genera Gluconobacter, such as Gluconobacter oxydans (U.S. Pat. Nos. 4,935,359; 4,960,695; 5,312,741; and 5,541,108), Pseudogluconobacter, such as Pseudogluconobacter saccharoketogenes (U.S. Pat. No. 4,877,735; European Patent No. 221 707), Pseudomonas, such as Pseudomonas sorbosoxidans (U.S. Pat. Nos. 4,933,289 and 4,892,823), and mixtures of microorganisms from these and other genera, such as Acetobacter, Bacillus, Serratia, Mycobacterium, and Streptomyces (U.S. Pat. Nos. 3,912,592; 3,907,639; and 3,234,105).
These processes, however, suffer from certain disadvantages that limit their usefulness for commercial production of 2-KLG. For example, the processes referenced above that employ G. oxydans also require the presence of an additional xe2x80x9chelperxe2x80x9d microbial strain, such as Bacillus megaterium, or commercially unattractive quantities of yeast or growth components derived from yeast in order to produce sufficiently high levels of 2-KLG for commercial use. Similarly, the processes that employ Pseudogluconobacter can require medium supplemented with expensive and unusual rare earth salts or the presence of a helper strain, such as B. megaterium, and/or the presence of yeast in order to achieve commercially suitable 2-KLG concentrations and efficient use of sorbose substrate. Other processes that employ Pseudomonas sorbosoxidans also include commercially unattractive quantities of yeast or yeast extract in the medium.
Other processes for the fermentative production of 2-KLG involving D-sorbitol as the starting material have also been developed. For example, 2-KLG can be produced from D-sorbitol with the aid of microorganisms of the genera Acetobacter, Bacterium or Pseudomonas which are capable of oxidizing D-sorbitol under aerobic conditions producing 2-KLG. However, according to this process, the yield of 2-KLG is low.
D-sorbitol as a starting material would be preferable to L-sorbose because it is less expensive. However, L-sorbose is more efficiently converted to 2-KLG. Therefore, a process that uses D-sorbitol as a starting material but provides L-sorbose for conversion combines the availability of D-sorbitol with the higher yield provided by L-sorbose. If part of the process is the conversion of D-sorbitol to L-sorbose, which is then converted to 2-KLG, then efficient production can be attained.
The conventional processes to produce 2-KLG from D-sorbitol via L-sorbose have been developed through two stage fermentation in which D-sorbitol is first converted to L-sorbose by one organism, and then L-sorbose is fed to another organism or mixed culture in a different medium to produce 2-KLG in a separate fermentor. A single stage fermentation using a single medium for a mixed culture to produce 2-KLG directly from D-sorbitol via L-sorbose in the same fermentor would provide a simpler and shorter fermentation with lower cost and higher yield for the production of 2-KLG from D-sorbitol versus the conventional method using two stage fermentation.
Accordingly, there still remains a need in the art for more efficient and economical 2-KLG-producing microorganism strains and processes.
The present invention provides microorganism strains and processes which efficiently produce 2-KLG from D-sorbitol via L-sorbose in a mixed culture. The invention provides a simpler and shorter fermentation with lower cost and higher yield for the production of 2-KLG from D-sorbitol versus the conventional method using two stage fermentation.
A further embodiment of the present invention is directed to strains for the production of 2-KLG in cooperation with helper strains.
Another embodiment of the present invention provides a process for the production of 2-KLG in which the fermentation medium contains soybean products.
An additional embodiment of the present invention is to provide the bacterial strains of the present invention transformed by a vector, and a method for the transformation of the bacterial strains by a vector.
These and other embodiments are accomplished by the present invention, which, in a first embodiment, is directed to a culture of a microorganism strain having the identifying characteristics of any of the microorganism strains NRRL B-30266, NRRL B-30265, NRRL B-30267 or NRRL B-30268, or mutants thereof derived from the strains.
Other features and advantages of the present invention will be set forth in the detailed description of preferred embodiments that follows, and in part will be apparent from the description or can be learned by practice of the invention. These features and advantages of the invention will be realized and attained by the methods particularly pointed out in the written description and claims hereof.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed.